Protein factors which specifically stimulate RNA polymerase II activity on native DNA templates at low ionic strength have been purified from calf thymus. The following steps were utilized in the initial purification: tissue homogenization, 40-80 percent ammonium sulfate cut, DEAE Sephadex and carboxymethylcellulose. Subsequent gel filtration (G-75) resolved two peaks of stimulatory activity. Each peak was further purified through SP-Sephadex chromotography. SDS polyacrylamide (10 percent) gels across the peak of activity of the lower molecular weight species (SF-B) revealed a single band with a molecular weight of 17,000. Gel filtration of SF-B under nondenaturing conditions on a calibrated G-75 Sephadex column gave a single broad peak between 17,000 and 34,000 molecular weight, suggesting a monomer-dimer equilibrium. Stokes radii calculations precluded a single dimer species, but not an asymmetric monomer (f/fo equals 1.3). The higher molecular weight species (SF-A) under nondenaturing conditions gave a single sharp peak with a molecular weight of 55,000. SDS gels of SF-A revealed several bands, but none less than 30,000, indicating that SF-A and SF-B are distinct entities. SF-B contained no detectable SV40 DNA (I) endonuclease, RNase H or RNase activities. BIBLIOGRAPHIC REFERENCES: Weil, P. A. and S. P. Blatti (1975) "Partial Purification and Properties of Calf Thymus Deoxyribonucleic Acid Dependent RNA Polymerase III." Biochemistry, 14, 1636. (Five reprints included). Weil, P. Anthony and Blatti, Stanley P (1976) "Hela Cell Deoxyribonucleic Acid Dependent RNA Polymerases: Function and Properties of the Class III Enzymes." Biochemistry, In press.